CHAPTER 2 PHARMACOGNOSY: MORPHOLOGICAL, ANATOMICAL AND PROXIMATE ANALYSIS OF LEAF, ROOT AND RHIZOME OF COSTUS IGNEUS (N.E.Br.) 2.1 INTRODUCTION The Costaceae was first raised to the rank of family by Nakai (1941). The family is one of the most distinctive and isolated members of the order Zingiberaceae (Dahlgren et al., 1985). Before the elevation to family status, Engler and Prantl (1930) recognized Costoideal as a subfamily under Zingiberaceae. Several anatomical and morphological features support this isolated position including well developed arial shoots with distinct, rigid and commonly branched stems. The leaves are inserted in a low spiral with divergences. The family Costaceae consists of four genera and approximately 200 species (Airy Shaw, 1973). The genus Costus is the largest in the family with about 150 species that are mainly tropical in distribution. In addition to its small size and distinctive morphology, Costus has a pan tropical distribution especially in the forest and savanna regions. Tomlinson (1956) presented some reasons for treating the Costaceae as a separate family based on a number of anatomical characters of the vegetative organs. Some of these diagnostic characters were summarized by Tomlinson (1956). These include the uniseriate filamentous, hairs isodiametric, irregular, or transversely extended thin walled epidermal cells, tetracytic stomata (stomata with subsidiary cells), slightly asymmetrical nature of guard cells, colours and continuous hypodermis, clearly differentiated cortex, well developed vascular bundle at each node restriction of silica to internal tissue close to vascular bundles, solitary bodies stellately spherical or drug like vessels restricted for root, etc. 21
(Edeoga, 1991). Apart from preliminary investigation of Tomlinson (1956) on the anatomy of some members of Costaceae no other information of costus igneus leaf, rhizome and root anatomy have been documented to the best of our knowledge. 2.2 MATERIALS AND METHODS 2.2.1 Collection of plant material Costus igneus were grown at Periyar Maniammai University nursery and were collected during the month of April (2008).The identification of the plant was confirmed and authenticated by Rapinant Herbarium, St. Joseph s College, Trichy, Tamilnadu, South India. Freshly collected samples were washed and used for study of organoleptic and microscopic characteristics. The dried powder samples were used for further analysis. All chemicals and reagents used in this study were analytical grade obtained from Hi Media, Qualigens and Loba Chemicals, available in India. 2.2.2 Morphological and Anatomical studies of Costus igneus 2.2.2.1 Specimens preparation Leaf, root and rhizome of Costus igneus obtained from living specimen of plant were fixed in FAE (1:1:18) (formalin 5ml + Acetic acid -5ml + 70% ethyl alcohol-90 ml) for 48-72 h. The specimens were dehydrated with graded series of tertiary butyl alcohol (TBA) as per the schedule given by Sass (1940). Infiltration of the specimens was carried by gradual addition of paraffin wax (melting point 58-60 C) until TBA solution attains super saturation. The specimens were cast into paraffin blocks. 2.2.2.2 Sectioning The paraffin embedded specimens were sectioned with the help of Rotary Microtome. The thickness of the sections was 10-12 µm. Dewaxing of the section was by customary procedure (Johansen, 1940). The sections were stained with Toluidine Blue as per the method published by O Brien et al., (1964). The dye rendered pink colour to the cellulose walls, blue to the lignified cells, dark green to 22
submarine, violet to the mucilage, blue to the protein bodies, etc., Wherever necessary sections were also stained with safranin and fast-green. For studying the stomatal morphology, venation pattern and trichome distribution, paradermal sections (sections taken parallel to the surface of leaf) as well as clearing of leaf with 5% sodium hydroxide or epidermal peeling by partial maceration employing Jeffrey s maceration fluid (Sass, 1940) were prepared. Glycerine mounted temporary preparations were made for macerated/cleared materials. Powdered materials of different parts were cleared with NaOH and then mounted in glycerine medium after staining. Different cell components were studied and measured. 2.2.2.3 Photomicrography Photographs of different magnifications were taken with Nikon lab photo 2 microscopic units. For normal observations bright field was used. For the study of crystals, starch grains and lignified cells polarized light was employed. Since these structures have birefringent property, under polarized light they appear bright against dark background. Magnification of the figures is indicated by the scale-bars. Descriptive terms of the anatomical features are as given in the standard plant anatomy text books. 2.2.3 Proximate Analysis The proximate analysis namely total ash content, acid insoluble ash, water soluble ash and moisture content were determined by the methods described by A.O.A.C (1990). The total carbohydrate and total protein were also determined by Anthrone method (Hedge and Hofreiter, 1962) and Lowry method (Lowery et al., 1953) respectively. 2.2.4 Statistical analysis The proximate analysis was carried out in triplicate for four separate experiments. The results were expressed as Mean ± Standard Deviation. 23
2.3 RESULTS 2.3.1 Morphological characters The Costus igneus is a rhizomatous shrub and penetrates through the tuberous rhizome. The rhizome is about 20-40cm grown, cylindrical, soft and fleshly with smooth pale brown surface. It is pleasantly aromatic. The leaves are green in color. Their length are about 15-25cm and narrow with several parallel equally thick veins. Tap root sub-cylindrical, wider at the top 28-50cm long, outer surface light brownish to pale dark brown (Figs. 2.1.1-2.1.3). 2.3.2 Anatomical studies of Costus igneus 2.3.2.1Anatomy of Rhizome The rhizome is circular with smooth and even surface. It has fairly distinct layer of epidermis, which consist of narrow oblong thin walled cells. Inner to the cortex is a wide cortical zone in radial plane. The cortical cells are circular wavy, thin walled, compact and possess wide spread structure. Randomly distributed in the cortex are small circular cortical vascular bundles (Figs. 2.3.1 and 2.3.2). The cortical bundles are collateral with a wide, angular fairly thick walled xylem elements and small cluster of phloem elements (Figs. 2.3.2 and 2. 4.1). Prominent clusters of xylem elements occur associated with the endodermoid layer (Fig. 2.3.2). The xylem elements are in tangential multiples; they are wide, angular, thick walled and measure 50µm in diameter. Phloem elements also occur as a thin sheath in-between the endodermis and xylem elements. The stele has an outer boundary of endodermoid layer, which is not well defined (Fig. 2.2.2). The stelar bundles are more numerous and scattered in the stele. They are also collateral and they are different in orientation (Fig. 2.4.2). The central (stelar) vascular bundles have wide, angular, thin walled metaxylem elements and three or four proto xylem elements (Fig. 2.4.2). The metaxylem elements are 70µm wide. Thick mass of phloem occurs adjacent to the meta xylem elements. The phloem elements are wide and prominent. 24
2.3.2.2 Anatomy of Leaf The leaf is thin with smooth even surfaces, isobilateral and has no difference between the upper and lower sides. The leaf has two layers of thin epidermal cells and four layers of wide, tangentially oblong thin walled mesophyll cells. Both epidermal layers have narrow, tangentially flat thin walled cells (Fig. 2.3.1); they are 10-20µm thick. The mesophyll cells are 100-140µm thick. These are prominent vascular bundles placed in the median part of the lamina. The bundles are collateral having wide mass of xylem elements and a small cluster of phloem. On the phloem end of the bundle, there is thick band of sclerenchyma cells are lignified (Figs. 2.3.2 and 2.3.3). The xylem elements are wide, angular and thick walled. The vascular bundles have no distinct bundle sheath cells. 25
Fig. 2.1.1 Costus igneus leaf Fig. 2.1.2 Costus igneus rhizome Fig. 2.1.3 Costus igneus root 26
Fig. 2.2.1 Rhizome outer sector (CB-Cortical vascular bundle; CO- Cortex) Fig. 2.2.2 Rhizome inner sector (EV-Endodermoid vascular bundle; SB-Stelar bundle;st- stele) 27
Fig. 2.2.3 Cortex with outer cortical bundle (CO-Cortex; Ep-Epidermis; OCBouter cortical bundle) Fig. 2.2.4 Inner cortical bundle with stelar vascular bundle (CO-Cortex; ICB-Inner cortical Bundle; OC-Oil contents; En-Endodermis; Ph- Phloem; St-Stele; X- Xylem Ph- Phloem) 28
Fig. 2.2.5 Endodermoid Vascular Bundle Magnified (CO-Cortex; End- Endodermoid layer; Ph- Phloem; X- Xylem) Fig. 2.2.6 Central stelar Vascular Bundle Magnified (GP-Ground parenchyma; SG- Starch grains; OC- Oil content; Ph- Phloem; X-Xylem) 29
Fig. 2.2.7 Cortical vascular bundle enlarged (MX- Metaxylem; Ph- Phloem) Fig. 2.2.8 Stelar vascular bundle enlarged (MX- Metaxylem; Ph- Phloem; PX- Proto xylem; SE- Sieve elements) 30
Fig. 2.3.1 T.S of leaf through lamina with vascular bundle (AbE- Abaxial epidermis; AdE- Adaxial epidermis; MT- Mesophyll tissue; VB- Vascular bundle) Fig. 2.3.2 Vascular bundle of the leaf enlarged (Ph- Phloem; SC- Sclerenchyma; X- Xylem) Fig. 2.3.3 Starch grains in the rhizome (under polarized light microscope) (SG- Starch grains) 31
Fig. 2.4.1 T. S of root a sector (CO- Cortex; X- Xylem; SPe- Stored periderm) Fig. 2.4.2 Same as above a portion enlarged (CO- Cortex; En- Endodermis; MX- Metaxylem; PC-Pericycle; Pi- Pith; Px-Protoxylem; SC-Sclerenchyma; SPe- Stored periderm; X-Xylem) Fig. 2.4.3 One vascular bundle magnified (MX- Metaxylem; En- Endodermis; PC-Pericycle; Px- Protoxylem;SC- Sclerenchyma; Ph- Phloem) 32
2.3.2.3 Anatomy of Root Thin roots have fairly wide superficial sequent periderm and narrow homogenous paranchymatous cortex. In olden thick root, the periderm is slightly wider and consists of polyhedral, randomly oriented, thin walled cells. The cortex is wider and parenchymatous with small, thin walled compact cells (Fig. 2.4.2). The periderm is 150µm wide and the cortex is 400µm wide. The stele has about 10 exarch xylem alternating with equal numbers of phloem strands (Fig. 2.4.1). The stele has distinct endodermis with cylindrical narrow cells; their inner and lateral walls have U-shaped thickenings. Inner to the endodermis is a narrow layer of hyaline cells, which are circular to rectangular in outline. The xylem strands are numerous and each strand has a wide circular, thick walled metaxylem elements and narrow proto xylem elements; the metaxylem is 60-70µm wide (Fig. 2.4.3); the proto xylem elements are 40 µm wide. A prominent mass of phloem occurs in between the xylem. No sclerenchyma tissue is seen in the stele. 2.3.3 Proximate analysis The result of fluorescent analysis, ash (total, acid soluble, water soluble) content, moister content, carbohydrate and protein of Costus igneus leaf, root and stem are represented in Tables 2.1, 2.2 and 2.3 respectively. 33
Table 2.1 Fluorescent analysis of leaf, stem and rhizome powder of Costus igneus under UV light and daylight S.No Chemical Leaf Rhizome Stem Test Day light UV light Day light UV light Day light UV light 1 Sample as Yellowish Light Greenish Light Brown such brown green brown brown Green 2 Extract with Dark Light Dark Dark Green aqueous brown brown brown Brown Brown NaOH 3 With Light Dark Light alcohol Brown Green Brown green brown green NaOH 4 With HCl Yellowish Light Light Light Light Light brown green brown green brown green 5 With 50% Dark Dark Light Dark HNO 3 brown green brown green Brown Green 6 Light With 50% Reddish Blackish Reddish Dark Blackish reddish H 2 SO 4 brown green brown green green brown 7 Methanol Yellowish Yellowish Dark Light Yellowish black black green brown brown Brown 8 With Light Light Yellowish Light Yellowish ammonia brown green brown green brown Brown 9 With I 2 Blue Blackish Blue Blackish Blue solution Brownish green Brownish green Brownish Black 34
Table 2.2 Behavior of leaf, rhizome and stem powder of Costus igneus with different chemical reagents Reagent Sample Name Colour / ppt Constituent Leaf Reddish brown Steroids Conc.Sulphuric Rhizome Reddish brown Steroids acid Stem Light reddish brown Steroids Leaf Dark Blackish brown Tannin Aqueous Ferric Rhizome Blackish brown Tannin chloride solution Stem Blackish brown Tannin Leaf Blue s brown Starch Iodine solution Rhizome Blue s brown Starch Stem Blue s brown Starch Leaf Dark yellowish brown Anthraquinone Ammonia Rhizome Yellowish brown Anthraquinone solution Stem Light Yellowish brown Anthraquinone Aqueous Leaf Yellowish brown Anthraquinone Potassium Rhizome Yellowish brown Anthraquinone hydroxide Solution (5%) Stem Light Yellowish brown Anthraquinone 35
Table 2.3 Proximate analysis of rhizome, stem and leaf of Costus igneus S.NO Parameters Costus igneus Root Stem Leaf 1 Total ash (%) 4.20±0.007 c 2.20±0.026 2.20±0.026 2 Acid insoluble (%) 0.20±0.020 0.13±0.073 0.17±0.022 3 Water soluble (%) 0.74±0.0267 1.00±0.027 1.00±0.027 4 5 Moisture (%) (After 1 hour) Moisture (%) (After 1.5 hour) 5.40±0.040 b 10.40±0.187 14.00±0.427 7.40±0.393 14.36±0.063 21.20±0.447 6 Carbohydrate (g/kg) 9.31±0.007 a 7.87±0.016 b, 11.20±0.107 7 Protein (g/kg) 2.78±0.002 a 1.60±0.016 a,, 3.30±0.06 The results were expressed as mean ± Standard deviation; Statistical significance variation were compared leaf Vs stem, rhizome; at a p<0.05, b p<0.01, c p<0.001, d p< 0.0001; rhizome Vs stem at, p<0.05, p<0.01,, p<0.001. 36
2.4 DISCUSSION The Costaceae plants were identified to have different morphological and anatomical characters of leaf, rhizome and root among them Costus igneus have a characteristic anatomy that could be used to distinguish it from other members of Costaceae. Costus igneus studied, however, appear to be a homogenous entity united by series of leaf, rhizome and root anatomical characters, including epidermal cells, mesophyll cells, the sclerenchyma cells, vascular bundle, cortex, xylem, phloem, starch grains, stelar bundle. These features of lamina, rhizome and root anatomy endorse. The finding of Oteng-Yeboah (1981) in the same species of Costus from West Africa coincides with our present study. Differences in vegetative anatomy among such members of Costaceae as Costus and Tapeinochilus (Tomlinson, 1956) have received some emphasis in phylogenetic discussion. The anatomical structures of leaf, rhizome and root of Costus igneus differentiated that of C. lucanusianus and C. afer that was studied by Edeoga and Okoli (1997). The leaf has two layers of thin epidermal cells and four layers of wide, tangentially oblong thin walled mesophyll cells. It is 300µm thick. Randomly distributed in the cortex are small circular cortical vascular bundles. The cortical bundles are collateral with a wide, angular fairly thick walled xylem elements and small cluster of phloem elements. Thin roots have fairly wide superficial sequent periderm and narrow homogenous paranchymatous cortex. The stele has about 10 exarch xylem alternating with equal number of phloem strands. No sclerenchyma tissue is seen in the stele. Tomlinson (1956) presented some reasons for treating Costaceae as a separate family from Zingiberaceae. The lamina, rhizome and root anatomy of these Costus species investigated seems to agree with the Tomlinson s (1956) reasons for supporting the raising of the genus to rank of family by Nakai (1941). Cytology, phytochemistry of Costus igneus studies helps to clearly differentiate Costus igneus from other Costaceae plant. Tomlinson (1956) outlined some diagnostic features of the leaf, stem, rhizome and root anatomy in the Zingiberales as a whole but did specify how these could be used in resolving critical systematic problem as in the presently investigated Costus igneus anatomical study. The proximate values shows that the protein content is relatively low but it can 37
contribute to the formation of hormones which controls a variety of body functions such as growth, repair and maintenance of body protein (Mau et al., 1999). The relatively high carbohydrate content can be used as energy source and also it is necessary in the digestion and assimilation of other foods. The moisture and ash content is useful in assessing the quality of grading the plant and also gives an idea of the amount of minerals present in the samples (Michael and David, 2002). At present investigation, Costus igneus have a characteristic anatomy and proximate analysis that could be used to distinguish it from other members of Costaceae. 38